Prevalence of mycorrhizae in host plants and rhizosphere soil: A biodiversity aspect

Plants roots are colonized by soil inhabitants known as arbuscular mycorrhizal fungi (AMF), which increase plant productivity, and enhance carbon storage in the soil. We found mycorrhizal vesicles, arbuscles, and mycelium in the root of more than 89% of the selected plants of University of Rajshahi campus, Bangladesh. The rate of their presence differed in plant to plant of a family and different families. The highest root colonization (98 ± 1.0%) was found to be present in Xanthium strumarium (Asteraceae). Mycorrhiza was not found in the root of Sphagneticola calendulacea (Asteraceae), Cestrun nocturnum (Solanaceae), Acacia nilotica and Acacia catechu (Mimosoidae), Rorippa nasturtium , Brassica oleracla var botrytis (Brasicaceae), Punica granatum (Lythraceae), Tecoma capensis (Bignoniacea), Spinacia oleracia (Chenopodiaceae), Chenopodium album (Goosefoot). Result of soil analysis reveals that the rhizospheric soils were deficient in nutrients which might be suitable for mycorrhizal symbiosis with plants. In the rhizospheric soils, 22 species of Glomus , Scutelospora , Gigaspora , Archaeospora , and Acullospora were found. We also found the genera ’ Glomus ’ dominance in the plant root and rhizospheric soil. So, it can be concluded that the highly colonized roots as well as spores can be used to prepare mycorrhizal inoculum for future purposes.


Introduction
Arbuscular mycorrhiza (AM) is the plant-fungal symbiosis that exists on the Earth [1,2]. Smith and Read [2] revealed that more than 90% of all plant species, from liverworts to angiosperms are involved in the mycorrhizal association [3]. In some cases, soil pH, soil phosphate (P) level, salinity vegetation, or the hydrologic condition of the soil have been found to be associated a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Sample collection
About 91 different plant species were selected randomly ( Table 1). The root samples along with rhizospheric soil were collected at a depth of 0-20 cm with the auger. Number of samples for each plant is one for soil sample and root pieces, and three plants are considered for each species.

Assessment of root colonization
Fixed root pieces were washed with 70% alcohol and then washed three times with distilled water. After that, root pieces were selected and cut into small segments (about 1 cm). Root segments were put in a beaker containing enough 10% KOH solution, covered, and heated at 90˚C in the water bath for 60 min. KOH was poured off and washed with distilled water three times. Root pieces were treated with alkaline H 2 O 2 for 20 min. at room temperature. Then, these were rinsed with distilled water three times and acidified with 1% HCl for 3 min. Root pieces were stained with trypan blue solution for 120-180 min., and subsequently, the root was de-stained at room temperature in lactoglycerol. After de-staining, these root Extraction, identification, and quantification of mycorrhizal spores. Collected soil samples were dried in the air, and 100 g of air-dried soil sample was taken in a bucket filled with ¾ th in tap water and mixed water properly, and left to settle down for about 5-10 min. The supernatant was decanted, and sucrose gradient centrifugation was done for 4 min. at 3000 rpm [11]. Spores were counted under a dissecting microscope, and spore densities (SD) were expressed as the number of spores per 100 g of soil. The isolated spores were mounted in polyvinyl lactoglycerol (PVLG). Morphological identification of spores up to species level was based on spore size, color, the thickness of the wall layers, and the subtending hyphae by the identification manual [http://schuessler.userweb.mwn.de/amphylo] and the website of the international collection of vesicular and AM fungi (http://invam.wvu.edu).

Soil analysis
Air-dried rhizospheric soil samples in three replicates for each plant were analyzed for their physical and chemical properties. The pH was determined (soil-water suspensions) with the help of a pH meter [12]. The texture was determined using 6% H 2 O 2 , 2N HCl, and 2N NaOH [13]. The moisture content was determined according to the conventional method. Organic matter (OM) was determined by the Walkley-Black acid digestion method. Phosphorus (extracted with 0.03M NH4F-0.02M HCl) was measured by molybdenum blue colorimetry method, potassium (K) by an ammonium acetate method using a flame photometer, and nitrogen (N) by the alkaline hydrolysis diffusion method. Available soil Boron and Zinc were determined in atomic spectrophotometer [14].

Statistical analysis
All experiments were conducted in triplicate. The data was analyzed by One-way analysis of variance (ANOVA), and the values of standard deviations were considered. The p-value (p < 0.05, < 0.001) was considered in determining significant difference.

Presence of AMF structure in roots
The plants of University of Rajshahi showed a well-colonized arbuscular mycorrhizal association. The occurrence of Mycorrhizal fungi in roots of plants has been determined on the basis of vesicles, arbuscular and hyphal formation (Fig 1).

Physio-chemical properties of rhizosphere soil
The degree to which mycorrhizal fungi enhance the nutrition and health of associated plants depends on many biotic and abiotic soil factors and other environmental factors that influence  Table 3 summarized the data on soil status, i.e. the physical and chemical properties. It may be mentioned that the status of soil means the suitability of soil conditions for various crop production. Soil quality influences mycorrhizal infection. Soil texture may affect plant responses to mycorrhizae. Soil strength and penetration resistance influence the rates at which water and nutrients flow or diffuse to the root surface. The clay, silt, and sand in experimental soils were varied from 8.00±0.15% to 18.1±0.31%, 27.8±0.51% to 38.0±0.32%, and 43.33±2.11% to 64.82 ±2.06% respectively. So, the observed soils were loamy soil. Loamy soil is suitable for growing most plant varieties.
It was revealed from the present data that the soil pH of the experimental plant area was alkaline, which might be associated with the natural colonization of an arbuscular mycorrhizal fungus in the roots of the plants examined. The pH range was indicated 7.30±0.13 to 8.10 ±0.20. Khan et al. (2004) reported that the soil pH of the Rajshahi region is high because of naturally alkaline, which is associated with occurring lime [15]. Soil pH is a commonly used index of plant root zone acidity and is crucial to many elements and microbial processes. Moisture influences soil resistance to root penetration, the geometry of different parts of the nutritional movement to root surface, and microorganism activity. The amount of moisture in soil was found to be 15.5±0.45% to 19.1±0.76% in Table 3. Phosphorus is one of the major nutrients for plant growth. It is the structural constituent of nucleotide, which is an energy carrier for all metabolic activities. It is essential for the constituent of the cell nucleus, cell division, and the development of meristematic tissue in the growing regions. The amount of phosphorus in experimental mycorrhizal soils was 15.48±0.51 ppm to 125.19±2.62 ppm. Nitrogen is an essential constituent of protein and, therefore, a constituent of all living cells. Nitrogen increases the proportion of water, and it also makes more giant cells with thinner cell walls.

PLOS ONE
Nitrogen is an essential constituent of protein and, therefore, a constituent of all living cells. Nitrogen increases the proportion of water, and it also makes more giant cells with thinner cell walls. Soil Nitrogen content was varied from 0.08±0.02 to 0.12±0.03%. Soil organic matter ranged from 1.39±0.05% to 2.11±0.08%. Potassium helps maintain cell permeability, aids in the translocation and composition of carbohydrates, and is essential for photosynthesis. Potassium keeps iron more mobile and increases the resistance of plants to a particular disease. The potassium levels of mycorrhizal rhizosphere soil were ranged between 0.16±0.02 cmol/kg to 1.09 ±0.09 cmol/kg. Zinc plays a central role in healthy plant metabolism and growth processes. It is needed in small quantities for the formation of auxin, chlorophyll, and cytochrome. It also has a role in forming enzymes and carbohydrates, regulating starches, and proper root development.
Zinc also helps plants assimilate to cold temperatures across the growing season. Zinc plays an essential role in mycorrhizal colonization and distribution. The level of zinc of mycorrhizal rhizosphere soil ranged from 2.28±0.06 ppm to 7.65±0.24 ppm. Boron plays a vital role in a diverse range of plant functions, including cell wall formation and stability, maintenance of structural and functional integrity of biological membranes, movement of sugar or energy into growing parts of plants, and pollination and seed set. Adequate Boron is also required for effective nitrogen fixation and nodulation in legume crops. Boron deficiency commonly results in empty pollen grains, poor pollen vitality, and fewer flowers per plant. Boron has a vital role in colonizing roots with mycorrhizal fungi, which contributes to root uptake of P. The Boron levels of mycorrhizal rhizosphere soil varied from 0.55±0.09 ppm to 1.61±0.18 ppm.
Results showed that mycorrhizal root colonization is positively correlated with number of spores. Correlation among mycorrhizal root colonization, spore numbers, and physiochemical properties of rhizospheric soils of 8 different plant species were summarized in Table 4.

Mycorrhizal spore density and diversity
The rhizospheric soils which were analyzed to determine physio-chemical properties were selected for isolation of mycorrhizal spores. Twenty-two different mycorrhizal spore populations were isolated. Nineteen isolated spores were identified based on morphological characteristics such as spore size, color, wall thickness, number of walls, types of walls and wall groupings, etc. Three spores could not be identified, which will be identified in future with 18s RNA technology. Identified spores belonged to the genera Glomus, Scutellospora, Gigaspora, Archaeospora and Acullospora mentioned in   Isolated spores were varied from 28.7±1.70 to 60.7±1.20 in number per 100 g of soil, as presented in Table 3. The highest spore number was observed in Codiaeum varicgatum, while that was lowest in Abelmoshcus esculentus.

Discussion
The study area, University of Rajshahi, Bangladesh has a tropical monsoon climate characterized by heavy seasonal rainfall, high temperatures, and high humidity. The mycorrhizal fungi were found to be present in nearly all of the tested plant species. The intensity varied in the plants of the same family and the plants of different families. Strzemska et al. [15] found that the root colonization in the plants of different families and the single-family plants differed [15]. The AM fungal structure in the root varied in the selected plants where vesicles, arbuscles, mycelium were present separately and in combination (Fig 1). Different vesicles were observed where some are oval and some are spherical in shape. Mycelia were present in most of the plants while arbuscles were observed in the roots of some plant species. The observed AMF structure was supported by Khanam et al. [16,17]. The frequent occurrence of vesicles in most plant species from the study sites showed the presence of VAM fungi belonging to the Glomineae. Plants of Brassicaceae Bignoniaceae, Goosefoot, and Chenopodiaceae were not colonized with mycorrhiza. These data are in line with earlier studies showing that these families lack functional mycorrhizae because of the presence of glucosinolates and their hydrolysis products, isothiocyanates, in and around their roots [18].
In rhizosphere of 8 different plant species, twenty two spores were isolated and nineteen spores were identified as species of Glomus, Scutelospora, Gigaspora, Archaeospora, and Acullospora (Fig 2) while three spores could not be identified. Among the identified genera, Glomus species was found more in number (Ten in twenty-two). Sporocarp of Glomus sinusum was observed which indicates that Glomus spores are grown in clusters. It might be a reason for getting more number of Glomus spores in the roots as shown in Fig 1. Glomus is the most common mycorrhizal species in Bangladesh's forests [19]. They speculated that Glomus' sporulation pattern could be the key to the taxon's rise to dominance. We found that the rhizosphere and roots of the same plant were found to contain a variety of species from different genera  (Figs 1 and 2). Plant phenology, root phenology, and root production all influence spore production patterns [20]. Every life history of a mycorrhizal fungus is influenced by plant roots. Spore germination, germination rate, the direction of germ tubes, hyphal branching recognition of the host root penetration establishment, intensity of colonization growth of hyphae into soils, and sporulation of the AM fungi were reported to be affected by the plant roots [21]. The roots of various plants produced a variety of organic chemicals and volatile compounds. Organic acids, ethanol and other volatile compounds could all influence the AM fungi's activity and life cycle in natural environments. Various factors, such as dense root systems with an abundance of fine roots, mycorrhizal fungi's ability to compete with other rhizosphere-dwelling organisms, seasons, soil moisture, soil type, and nutrient levels, have been found to have an impact on spore numbers, activity, and other traits [21].

PLOS ONE
AM fungal spore number was found to be increased with increase in root colonization. This result is consistent with those of the previous reports [22,23]. However, Fontenla et al. [24] have demonstrated that when the number of spores was high, the frequency of colonization decreased [24]. It has been shown that there is no significant relationship between AM colonization and spore population [25]. It might be due to the different gradients by soil and the strong effect of plant factors on the formation, function, and adaptation of the fungus to the respective soil conditions. Mycorrhizal colonization was found to be possible due to the presence of moisture [26]. Roots were found to have arbuscles when examined under a microscope. Moisture may be a factor for the occurrence of arbuscles. There are several factors that can influence the growth, sporulation, and community structure of AM fungi in the soil [27,28]. The alkaline soil in the experimental area could be linked to the natural colonization of AM fungi [26]. The extraradical proliferation of hyphae may be aided by organic matter, which increases spore production [29,30]. A high level of soil phosphorus generally inhibits mycorrhizal infection [31][32][33][34] which might be the reason for current prevalence of mycorrhiza in Abelmoshcus esculentus. The potassium concentration might be suitable for mycorrhizal colonization in Bangladesh [35]. The zinc concentration in the soil might be suitable for mycorrhizal root colonization [36]. Mycorrhizal root colonization also affects the zinc nutrition of the crop [37] and is inurn affected by zinc status of the soil [38], climate changes [39,40] and the presence of organic fertilizers and rhizobia [41,42].
However, chemical analysis showed that the rhizospheric soils were deficient in nutrients, especially C, N. Nutrient deficiency might be responsible for the variation in their pattern of production and colonization. By considering all the facts mentioned above, it can be said that the ecological condition of the study area favored diverse mycorrhizal prevalence and their colonization in plant roots.

Conclusion
This study reveals that in University of Rajshahi, Bangladesh, plant species respond to mycorrhizal association where about 89% plant species are involved in mycorrhizal association. Species of Acaulospora, Gigaspora, Glomus, Scutelospora, and Archaeospora was observed in the selected rhizospheric soils. Three spores could not be identified which will be confirmed with 18s RNA technology. So, it can be concluded that the highly colonized roots as well as spores can be used in inoculum production on crop of Bangladesh.